Oligonucleotides and their analogs are routinely used in many diagnostic and research applications, as probes, primers, linkers, adaptors and antisense oligonucleotides. Antisense oligonucleotides have been used routinely in research to study the functions of gene products, i.e. proteins, by modulating the expression thereof. These oligonucleotides are designed to bind in a specific fashion to a particular mRNA sequence by hybridization (i.e., oligonucleotides that are specifically hybridizable with a target mRNA). Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA by any of a number of mechanisms, i.e., to interfere with translation reactions by which proteins coded by the mRNA are produced or initiate RNase H degradation of the mRNA. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences allows the study of functions of certain genes.
The specificity of antisense oligonucleotides and their analogs suggests that they might be useful therapeutically. Their mechanism of action should limit side effects while increasing specificity. Presently, there are numerous antisense oligonucleotides in clinical trials against a wide range of targets and diseases and recently the first antisense oligonucleotide was approved by the FDA for marketing.
Applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities. This is especially true for their use as commercially available pharmaceutical drugs. The large-scale synthesis of oligonucleotides on an economic scale presents different challenges than those in synthesis of small amounts for research.
Synthesis of oligonucleotides can be accomplished using both solution phase and solid phase methods. A general review of solid-phase versus solution-phase oligonucleotide synthesis is given in the background section of Urdea et al. U.S. Pat. No. 4,517,338, entitled "Multiple Reactor System And Method For Oligonucleotide Synthesis". Oligonucleotide synthesis via solution phase can be accomplished with several coupling mechanisms.
One such solution phase preparation utilizes phosphorus triesters. Yau, E. K. el al., Tetrahedron Letters, 1990, 31, 1953, report the use of phosphorous triesters to prepare thymidine dinucleoside and thymidine dinucleotide phosphorodithioates. However, solution phase chemistry requires purification after each internucleotide coupling, which is labor-intensive and time-consuming.
Further details of methods useful for preparing oligonucleotides may be found in Sekine, M. et. al., J. Org. Chem., 1979, 44, 2325; Dahl, O., Sulfer Reports, 1991, 11, 167-192; Kresse, J. et al., Nucleic Acids Res., 1975, 2, 1-9; Eckstein, F., Ann. Rev. Biochem., 1985, 54, 367-402; and Yau, E. K. U.S. Pat. No. 5,210,264.
The current method of choice for the preparation of naturally-occurring oligonucleotides, as well as oligonucleotides with modified internucleotide linkages such as phosphorothioate and phosphoro-dithioate oligonucleotides, is via solid-phase synthesis. Solid-phase synthesis involves the attachment of a nucleoside to a solid support, such as a polymer support, and the addition of a second nucleotide onto the support-bound nucleotide. Further nucleotides are added, thus forming an oligonucleotide which is bound to a solid support. The oligonucleotide can then be cleaved from the solid support when synthesis of the desired length and sequence of oligonucleotide is achieved.
Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide chain. Typically, a first nucleoside, having protecting groups on any exocyclic amine functionalities present, is attached to an appropriate solid support. Activated phosphorus compounds, typically nucleotide phosphoramidites, also bearing appropriate protecting groups, are added step-wise to elongate the growing oligonucleotide. The activated phosphorus compounds are reacted with the growing oligonucleotide using "fluidized bed" technology to mix the reagents.
A number of solid-phase synthesizers are available commercially. These are suitable for preparing relatively small quantities of oligonucleotides, i.e., from about the micromolar (.mu.mol) to millimolar (mmol) range. They typically are not amenable to the preparation of the larger quantities of oligonucleotides necessary for biophysical studies, pre-clinical and clinical trials and commercial production due to the high cost of reagents.
Instruments for large-scale solid phase synthesis of oligonucleotides are also commercially available, for example, the Pharmacia OligoPilot II and Milligen/Biosearch 8800. Although the process used by these machines is well understood, they require use of expensive reagents. Given the vast amounts of oligonucleotide syntheses performed for research use and for large scale manufacture pursuant to clinical trials, any waste of these expensive reagents is a significant economic problem.
One reagent used in large quantities is acetonitrile, which is used in multiple washing steps, as a solvent for an activator during the coupling step of the phosphoramidite method, and as a solvent for phosphoramidites, capping solution and an oxidation reagent. It is accepted dogma by those skilled in the art that "low water content" acetonitrile, i.e. having a water content less than 30 ppm, is mandatory for oligonucleotide synthesis. See, in general, Gait, M. S., Oligonucleotide Synthesis A Practical Approach, IRL Press 1985, p. 18-19. It is believed that using acetonitrile with a greater water content during any of the synthesis steps results in sub-optimal yields. The presence of water in the system is thought to interfere with the coupling reaction. Consequently, costly low water content acetonitrile is universally used for oligonucleotide synthesis. "Reagent grade" acetonitrile, acetonitrile having a water content higher than 30 ppm is available, but the process of removing water and other impurities from such reagent grade acetonitrile is costly and lengthy. See, U.S. Pat. No. 5,440,068 for a review of the art.
Thus there remains a need for improved and more economical processes for oligonucleotide synthesis.